Diaphorase is an enzyme catalyzing the reduction of a pigment with nicotinamide adenine dinucleotide (hereinafter abbreviated as NADH) or nicotinamide adenine dinucleotide phosphate (hereinafter abbreviated as NADPH) (the abbreviation NAD(P)H will hereinafter be used to denote either one of NADH and NADPH).
Measurements of diaphorase activity have generally been made by colorimetrically analysis of changes in absorbance which accompany reduction of pigments. Pigments reducible in the presence of diaphorase include, e.g., dichlorophenolindophenol (hereinafter abbreviated as DCIP) and NBT.
In using DCIP as a reducible pigment (substrate), DCIP has an absorption at a wavelength of 600 nm and is reduced with NAD(P)H to reduced-DCIP having no absorption at 600 nm, and the decrease in absorbance at 600 nm is thus used to assay diaphorase activity (Biochem. J., Vol. 191, pp. 457-465 (1980)). In this method, it is necessary to start measurement with a pigment solution having a high absorbance. Therefore, the absorbance at 600 nm at the start of measurement is not constant, and varies for each measurment. Further, even with no enzyme present in the solution, DCIP is reduced with NAD(P)H through non-enzymatic reaction which also causes reduction in absorbance at 600 nm.
Alternatively, measurement of diaphorase activity using NBT can be effected by measuring the increase in absorbance at 550 nm due to the formation of diformazan by the reduction of NBT. Accordingly, any pigment solution should have zero absorbance at 550 nm at the start of measurement. A method using NBT as a substrate therefore achieves more accurate diaphorase assay results than one using DCIP as a substrate. For this reason, when diaphorase is used when sensitivity and accuracy are of importance, for example, in an enzyme immunoassay, NBT is used as a substrate more widely than DCIP (see, e.g., JP-A-60-214900 and JP-A-60-58097 (the term "JP-A" as used herein means an unexamined published Japanese patent application)).
In spite of the above-described advantages, NBT is also known to suffer from problems, such as being non-enzymatically converted to diformazan in the presence of NAD(P)H, causing the absorbance at 550 nm to gradually increase. Therefore, when measuring diaphorase activity using NBT, a solution containing diaphorase and a blank solution containing no diaphorase are assayed to determine the increase of absorbance in each solution, and the increase in absorbance of the blank solution is substracted from that of the NBT solution. That is, the measured value is the sum of the increase in absorbance arising from (A) the enzyme reaction of diaphorase and (B) the non-enzymatic action of the blank so that subtraction of the blank value from the measured value gives more accurate and precise diaphorase activity. However, such a method of measurement, when a small amount of diaphorase is assayed, suffers from the additional problem that the ratio of the blank value to the true value attributed to enzyme reaction becomes high, so that the blank value can be many fold higher than that of the enzyme activity value, thus making it difficult to provide an accurate measurement of diaphorase activity even when a blank solution is used.